Response to interferon of patients infected with HCV has been variable. Recent studies suggested a region, termed IFN sensitivity determining region (ISDR) in the HCV NS5A gene, that are associated with resistance to interferon. The NS5A has also been shown to be a phosphoprotein and probably plays an important role in viral replication and viral-host interaction. Because of the functional importance of NS5A, our laboratory is conducting experiments to characterize its functions and identify cellular factors that are the functional targets of this HCV gene product. Using the yeast two hybrid system, 13 independent clones that interact specifically with NS5A have been identified. Many of these clones encode proteins with SH3 and/or SH3 binding domains and some of them are known genes with putative signal transduction functions. Interactions of these clones with NS5A were also confirmed in the GST-fusion in vitro binding assay. It is interesting to note that several proline-rich sequences (similar to SH3 binding domain) are present and flank the ISDR region in NS5A. Experiments are under way to address the functional significance of these interactions. Effort is also being initiated to evaluate the clinical significance of sequence variations in NS5A. Detailed characterization of this virus-cell interaction and correlation to clinical disease may contribute to our understanding of HCV replication and mechanisms of hepatocellular injury.